The incidence of serious bacterial and fungal infections is increasing despite remarkable advances in antibiotic therapy. Each year there are more than 40 million hospitalizations in the United States, and about 2 million patients acquire nosocomial infections, 50 to 60% of which involve antibiotic-resistant bacteria. The number of deaths related to nosocomial disease is estimated at 60,000-70,000 annually in the USA and up to 10.000 in Germany. Whereas resistant Gram-negative bacteria were a major problem in the 1970s, the 1990s had seen a climb in number of incidents with multi-drug resistant Gram-positive strains. Currently, the rapid emergence of resistant strains involves both Gram-positive and Gram-negative pathogens. Resistances developed first in species in which single mutations were sufficient to cause clinically important levels, such as Staphylococcus aureus and Pseudomonas aeruginosa, followed by bacteria in which multiple mutations are required, such as E. coli and Neisseria gonorrhoeae. This is mainly due to the broad use of fluoroquinolones. Important causes of Gram-negative resistance include extended spectrum beta lactamases in Escherichia coli and Klebsiella pneumoniae. Almost half of the clinical strains of Haemophilus ducreyi, the causative agent of chancroid, carries genes to confer resistance to amoxicillin, ampicillin and a series of other β-lactams. Likewise, for Salmonella enterica serovar. Typhimurium, resistance towards tetracyclines has increased from zero in 1948 to a 98% level in 1998.
This necessitates a continuing search for novel antibiotics. Inducible antibacterial peptides represent a field of study where contemporary biochemistry, immunology and drug design converge. Peptide antibiotics, ranging in size from 13 to more than a hundred amino acid residues, have been isolated from plants, animals and microbes (Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13: 61-92). The overwhelming majority of antibacterial peptides, including the well-studied defensins, cecropins and magainins, function through a “lytic/ionophoric” mechanism. Common theme among all “lytic” peptides is a permeabilizing effect on bacterial cytoplasmic membranes. Peptides acting directly on the bacterial membrane often have also toxic effects on mammalian membranes at higher concentrations, which limits their potential as future drugs. When prolines are inserted into the sequences of α-helical antimicrobial peptides, the peptides' ability to permeabilize the cytoplasmic membrane of E. coli decreases substantially as the function of the number of proline residues incorporated. In this regard, it is intriguing that some of the most active native antibacterial peptides, at least against selected Gram-negative pathogens, belong to the proline-rich peptide family.
Proline-rich antimicrobial peptides, including apidaecin, originally isolated from insects, have been postulated to overcome said shortcuts. Furthermore, modification of specific peptide bonds within the apidaecin has been modified at different positions in order to obtain a peptide being more resistant to cleavage, e.g. by proteases (WO-A 2009/013262). The inventors furthermore found that modification at different positions of an artificial peptide (oncocin; SEQ ID NO. 1)-said peptide is based on the sequence of Oncopeltus fasciatus antibacterial peptide 4 (SEQ ID NO. 5)-results in an increased stability against proteases and an additional dramatically enhanced antimicrobial activity (PCT/EP2010/051072; not yet published). However, there is still a need for antimicrobial peptides with an increased stability and enhanced antimicrobial activity.
TABLE 1SEQ IDPeptideSpeciesSequence NO.Ref.OncopeltusOncopeltusVDKPPYLPRP(X/P)PPRRIYN(NR)5[1]antibacterialfasciatuspeptide 4 OncocinartificialVDKPPYLPRPRPPRRIYNR-NH21[2]
[1] Schneider M & Dorn A. Differential infectivity of two pseudomonas species and the immune response in the milkweed bug, Oncopeltus fasciatus (Insecta: Hemiptera). Journal of Invertebrate Pathology 78: 135-40, 2001
[2] (PCT/EP2010/051072; not yet published)
The inventors now unexpectedly found that the substitution of isoleucine at position 16 of oncocin (SEQ ID NO. 1) leads to peptides or peptide derivatives which exhibit a dramatically increased stability and antimicrobial activity when compared with the Oncopeltus antibacterial peptide 4 (SEQ ID NO. 5).